Clarias Batrachus, C
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J. Asiat. Soc. Bangladesh, Sci. 41(1): 51-58, June 2015 DIFFERENTIATION OF CLARIAS BATRACHUS, C. GARIEPINUS AND HETEROPNEUSTES FOSSILIS BY PCR-SEQUENCING OF MITOCHONDRIAL 16S rRNA GENE MOHAMMAD SHAMIMUL ALAM1, HAWA JAHAN2, ROWSHAN ARA BEGUM2, REZA M. SHAHJAHAN2* 1Department of Botany Dhaka University Dhaka, Dhaka-1000 2Department of Zoology, University of Dhaka, Dhaka 1000, Bangladesh Abstract Heteropneustes fossilis, Clarias batrachus and C. gariepinus are three major catfishes of ecological and economic importance. Identification of these fish species becomes a problem when the usual external morphological features of the fish are lost or removed, such as in canned fish. Also, newly hatched fish larva is often difficult to identify. PCR- sequencing provides accurate alternative means of identification of individuals at species level. So, 16S rRNA genes of three locally collected catfishes were sequenced after PCR amplification and compared with the same gene sequences available from other geographical regions. Multiple sequence alignment of the 16S rRNA gene fragments of the catfish species has revealed polymorphic sites which can be used to differentiate these three species from one another and will provide valuable insight in choosing appropriate restriction enzymes for PCR-RFLP based identification in future. Key words: PCR-sequencing, 16S rRNA, Heteropneustes, Clarias, Species identification Introduction Catfishes of the genus Heteropneustes and Clarias (Siluriformes: Heteropneustidae, Clariidae) are commercially and ecologically important groups of freshwater fishes. These closely related species share more or less conservative external morphology and are difficult to identify when usual external features are missing. For instance, during fish processing into portions of flesh, such as in canned fish, species identification could be impossible due to loss of identifying characteristics (Quinteiro et al. 1998). Also, newly hatched fish larvae are often difficult to differentiate based on morphology and alternative method is in use (Lindstrom 1999). In addition, species complexes are difficult to identify based on morphology alone. Molecular techniques, especially DNA-based species identification techniques are better alternatives to morphology-based ones (Teletchea 2009). Analysis of polymerase chain reaction (PCR) amplified DNA fragments can provide an accurate alternative means of identification of individuals to genes or species level (Hubalkova et al. 2008, Jans et al. 1Corresponding author: E-mail: [email protected] 52 Alam et al. 2012, Kochzius et al. 2010, Lakra et al. 2011, Scheidegger et al. 2009 and Teletchea 2009). Among many PCR-based approaches, PCR-sequencing is found to provide highest amount of information and has been widely used now-a-days (Hajibabaei et al. 2007, Rehbein 2013, Zeng et al. 2013 and Zhuang et al. 2013). PCR-RFLP (Restriction Fragment Length Polymorphism) is another good choice for differentiation of species with known sequence. PCR-RFLP of different genes was demonstrated to detect inter- and intra-specific variations in several animals (Di Finizio et al. 2007 and Duduk et al. 2013). PCR-RFLP analysis is faster and more cost effective. Mitochondrial DNA (mtDNA) is maternally inherited and does not go through recombination event. Single nucleotide polymorphism in the mitochondrial genome increase the power of discrimination among the individuals (Coble et al. 2004). mtDNA genetic markers, such as Cytochrome oxidase I (COI), 16S and 12S rRNA genes have been widely used as tools to distinguish within and among species (Di Finizio et al. 2007, Klossa et al. 2002 and Wolf et al. 2000). Recently, 16S rRNA gene sequences are reported to be particularly useful in identifying different animal species (Sarri et al. 2014). In this background, the main objectives of the present investigations are (1) to compare the 16S rRNA gene sequences of three catfishes from Bangladesh with the existing sequences of those in the GenBank database, (2) to compare sequences among the three selected catfishes and (3) to explore the possibility of identifying these three species using PCR-RFLP on 16S rRNA gene sequence. Materials and Methods Sample Collection and DNA Extraction: H. fossilis (locally known as Shing), C. batrachus (Magur) and C. gariepinus (African Magur) were collected from local fish markets (Dhaka, Bangladesh). DNA was extracted from 50 mg muscle tissue of each fish species following a standard method (Doyle and Doyle 1990) with modifications. CTAB extraction buffer (1M 10 ml Tris-HCl [pH 8], 0.5M 4ml Sodium-EDTA [pH 8], 8.18 g NaCl, 2g CTAB, dH₂O) and 10 µl of 20 mg/mL proteinase K were added with minced meat sample. The mixture was incubated at 55°C for 2 h. After digestion, the samples were centrifuged at 13000 rpm for 5 min and equal volume of Phenol: Chloroform was added to the supernatant. Then, DNA was precipitated with ethanol and dissolved in distilled water. PCR amplification and Sequencing: Universal primers for PCR of the 16S rRNA gene, 16Sar-L (5’-CCGGTCTGAAAAAAACAT) and 16Sar-H (5’-CCGGTCTGAA CTCAG ATCACGT) have been utilized (Palumbi 1996). Standard PCR was performed using PCR Master Mix (Promega). Temperature regimes for PCR amplifications were as follows – initial denaturation at 95°C for 3 min, followed by 30 cycles of 95°C for 30 s, 55°C for 30s and 72°C on the last cycle. PCR products were run in an agarose gel containing ethidium bromide. The electrophoresis was performed at 90 V for 30 min and Differentiation of Clarias batrachus 53 DNA bands were visualized on a UV transilluminator and photographed. Then, PCR amplicons were purified using a purification kit (FavorGen) and sequenced. Bioinformatics: 16S rRNA gene sequences of experimental fish species and existing sequences of related fish species were collected from GenBank database (NCBI). Suitable portion of each sequence was taken for further observation. A multiple sequence alignment was performed using ClustalW software to observe the polymorphic sites present among the sequences. Restriction endo-nuclease cutting sites were searched in the sequences of the 16S rRNA fragment analyzed further. ‘Serial Cloner’ software was used to observe restriction sites. Results and Discussion Comparison of 16S rRNA gene sequence of three catfishes from Bangladesh with the available sequences from GenBank database: Partial sequences of 16S rRNA genes (5 Clarias spp. and 2 Heteropneustes spp.) were retrieved from GenBank database (Table 1). Suitable portion of DNA sequence (560 bp) was analyzed further. A multiple sequence alignment was done by SeaView to compare the sequences (Gouy et al. 2010) (Fig. 1). After comparing these sequences in different individuals, 55 out of 560 nucleotide bases of the sequence were found polymorphic. Among these polymorphic sites, nine were selected as positions with diagnostic value at genus level (Table 2). In seven positions (137, 219, 325, 367, 439, 443, 463) genus Heteropneustes and genus Clarias show nucleotide variations. In another two positions (242, 328), Clarias has particular nucleotide bases though deletion was observed in Heteropneustes. These diagnostic positions were selected using the criteria that these positions did not show intra-specific variability. In some positions, inter-species variations were also observed. 16S rRNA gene sequence of H. fossilis differs from that of H. microps in positions 34, 181, 241, 261, 313, 326, 331, 336, 338, 351, 361. Besides, H. fossilis sequence from Bangladesh differs from that of other geographical regions in the positions 271 and 377. Mitochondrial DNA diversity due to geographical differences has been reported elsewhere (Linares et al. 2009). Table 1. List of 16S rRNA sequences used in this study. Scientific name Common name GenBank Accession Collected Clarias batrachus Walking Catfish (Magur) KF997532.1*Numbers Bangladeshfrom C. batrachus Walking Catfish JQ699193 India C. gariepinus North African Catfish (African KJ819942* Bangladesh C. gariepinus NorthMagur) African Catfish JQ699188 India C. dussumieri - JQ699198.1 India C. fuscus White spotted Clarias JN020056.1 China C. gabonensis - JX899749.1 Gabon Heteropneustes Stinging Catfish (Shing) KJ819943* Bangladesh H.fossilis fossilis Stinging Catfish FN677932 India H. fossilis Stinging Catfish GQ411079 India H. microps Stinging Catfish FJ432686 India *Sequences submitted to GenBank from this study. C. dussumieri 54 Alam et al. Sequence comparison among three selected catfishes: Fragments of mtDNA of three selected catfishes (Clarias batrachus, C. gariepinus and Heteropneustes fossilis) were sequenced. The GenBank accession numbers are KF997532.1, KJ819942 and KJ819943, respectively. A multiple sequence alignment was performed by SeaView (Gouy et al. 2010). Twenty nine out of 560 bases of the sequence were found polymorphic. Among the polymorphic sites, 15 have diagnostic value at genus level, i.e, genus Clarias and genus Heteropneustes can be differentiated by these 15 nucleotide variations (Table 3). Comparing the sequences, polymorphic sites were also observed between two species belonging to the genus Clarias. Fifteen positions were found which have diagnostic value at species level (Table 4). Two species of Clarias (C. batrachus and C. gariepinus) can be differentiated by this nucleotide variation. Fig. 1. Multiple sequence alignment of 16S rRNA gene fragments of Clarias spp. and Heteropneustes spp. Some representative polymorphic sites are indicated by the nucleotide position numbers of Clarias